Nuclear transport is essential to a number of biological processes including gene expression and cell division, as well as to viral replication, tumorigenesis and tumor cell proliferation. The mechanism of nuclear transport has only recently been characterized in detail and has been shown to involve a number of discrete steps. Proteins that are destined to be transported into the nucleus contain within their amino acid sequence a short stretch of amino acids termed a nuclear localization sequence ("NLS"). These sequences are generally basic in nature, however, there has been no consensus sequence identified. Thus, there is a wide variety of these sequences that appear to be specific for particular proteins.
Within the cell, these NLSs may be either masked or unmasked by accessory proteins or by conformational changes within the NLS-containing protein. An NLS may be masked because it is buried in the core of the protein and not exposed on the surface of the protein. Unmasking of NLSs, and nuclear translocation of cytoplasmic proteins may be triggered by phosphorylation, dephosphorylation, proteolytic digestion, subunit association or dissociation of an inhibitory subunit, or the like. Accordingly, the masking and unmasking of NLSs provides a mechanism by which the transport of these cytoplasmic proteins into the nucleus may be regulated.
Nuclear translocation of transcription factors requires the presence of an unmasked or activated NLS in the nucleus-targeted protein. The binding of certain ligands to cell surface receptors activates the nuclear translocation of cytoplasmic transcription factors. Once in the nucleus, these transcription factors exert gene expression modulatory activity.
NF-.kappa.B is a ubiquitous transcription factor found in various levels and states of activation in different cell types. NF-.kappa.B is composed of several different subunits including p65, p50, c-rel p52 and p105. Recent studies suggest that distinct NF-.kappa.B complexes contribute to the regulatory control of gene transcription. The function and regulation of NF-.kappa.B has been most well-characterized in lymphocytic cells. In these cells, there is a wide variety of target genes, e.g., immunoregulatory genes, that are regulated by NF-.kappa.B including .kappa. Ig light chains. Such genes include those that encode the interleukin-2" ("IL-2.alpha.") receptor, interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), tumor necrosis factor-.alpha. ("TNF-.alpha."), and the like.
In unstimulated cells, a major form of NF-.kappa.B is a heterodimer of p50 and p65 (RelA) subunits. Nonactive NF-.kappa.B is retained in the cytoplasm as an inactive complex by inhibitory proteins such as I.kappa.B.alpha., .beta. and .gamma.. When cells are appropriately stimulated, e.g., by a proinflammatory stimulus such as a cytokine, the I.kappa.Bs are degraded, thereby releasing free NF-.kappa.B dimers, which translocate to the nucleus and activate target genes, e.g., lymphokine genes and other immunoregulatory genes. This response is transient and is terminated through delayed NF-.kappa.B-mediated I.kappa.B.alpha. induction.
Recently it has been demonstrated that glucocorticoids exert their immunosuppressive activity by inhibiting NF-.kappa.B nuclear translocation. Scheinman et al. (1995) Science 270:283-286 and Auphan et al. (1995) Science 270:286-290 independently demonstrated that the inhibition is mediated by an increase in the induction by glucocorticoids of I.kappa.B inhibitory proteins. These investigators proposed that inhibitors of NF-.kappa.B may be useful immunosuppressive and anti-inflammatory agents. Such an NF-.kappa.B nuclear translocation inhibitor, comprising an NLS from the p50 subunit of NF-.kappa.B attached to a membrane-permeable polypeptide motif, was described in Lin et al. (1995) J Biol. Chem. 270:14255-14258.
Nuclear translocation of proteins other than endogenous transcription factors and other cytoplasmic proteins also depends on the presence of an activated or unmasked NLS. For example, nuclear translocation of the retroviral preintegration complex is a crucial step in human immunodeficiency virus type-1 ("HIV-1 ") replication in nondividing cells such as monocytes and growth-arrested T cells. Such translocation is dependent on the presence of an NLS in the N-terminal portion of HIV matrix antigen ("MA") p 17. Indeed, the HIV-1 enhancer contains tandem binding sites for NF-.kappa.B that can be essential for virus replication (Ross et al. (I 991) J Virol. 65:4350-4358; Parrott et al. (1991) J Virol 65:1414-1419). Nuclear translocation of the HIV-1 preintegration complex can be partially inhibited by an excess of the SV40 large T antigen NLS (Gulizia et al. (1994) J Virol. 68:2021-2025). Furthermore, Dubrovsky et al. (1995) Molecular Med. 2:217-230 reported that a series of compounds capable of binding to and reacting with the HIV-1 MA p17 NLS inhibit HIV-1 replication in human monocytes.
In addition, tumorigenesis and tumor cell proliferation are regulated by the expression of oncoproteins, many of which are cytoplasmic transcription factors that are translocated into the nucleus by virtue of the presence of an NLS. Miller et al. (1996) J Cell Biochemistry 60:560.
Prior to Applicants invention, those in the art failed to demonstrate the synergism between peptide inhibitors of nuclear translocation and other imunosuppressants, such as cyclosporin. Buelow et al. (1995) Transplantation 59:455 demonstrated that therapy with a small synthetic peptide derived from the .alpha.1 helix of an HLA class I molecule (called the ALLOTRAP peptide), combined with a subtherapeutic dose of cyclosporin, led to the prolonged survival of allografts. The ALLOTRAP peptide, however, does not inhibit nuclear translocation of protein, an essential element of Applicants invention.
Many immunosuppressants are known in the art to be useful in treating autoimmune disease and in preventing transplant rejection. Examples of known immunosuppressants useful in compositions of the present invention are cyclosporin A, mycophenolate mofetil, rapamycin, FK506, and steroids. Compositions of the present invention comprising at least one peptide inhibitor of nuclear translocation of a protein also comprise at least one immunosuppressant. Together, the peptide inhibitor and immunosuppressant work synergistically to provide better immune suppression than either treatment alone.
Accordingly, inhibitors of nuclear translocation of cytoplasmic proteins would be useful as gene expression modulating agents, immunoregulatory agents, antiviral agents, antitumor agents, and the like. Such inhibitors, in combination with other immunosuppressant compounds such as cyclosporin A, would provide useful compositions to regulate immune responses (e.g., prevent transplant rejection).